The present invention relates to a method and apparatus for effecting rapid thermal cycling of samples. The invention is particularly useful in a process called xe2x80x9cPolymerase Chain Reactionxe2x80x9d, or PCR, in a biological process called Cyclic DNA Amplification, and is therefore described below particularly with respect to this application, but it will be appreciated that the invention could advantageously be used in other applications involving the heating and/or cooling of samples.
The PCR process involved in Cyclic DNA Amplification requires rapidly varying the temperature of a plurality of samples repeatedly through predetermined temperature cycles in a precisely controlled manner. Existing thermal cyclers can generally be divided into two main categories: (a) cyclers based on thermal blocks into which the samples are introduced and which are heated and cooled by Peltier elements; and (b) cyclers based on the circulation of air, water or other fluids.
In the case of thermal-block cyclers using Peltier elements, the large thermal mass of the block causes the heating/cooling process to be slow. A typical 30/cycle PCR process based on this technique generally takes about 2-2.5 hrs to complete. Another disadvantage of the block-cyclers is the difficulty in pulling the sample holders (e.g., microtiter plates) out of the block at the end of the process because of the deformation of the sample holders, usually plastic wells, caused by the temperature variations. Such deformations make it difficult to interface the cycler with robots which are generally needed for high-throughput processing.
Thermal cyclers using water baths have also been used for this purpose, but the high thermal mass of the water also extends the time required for a complete heating/cooling cycle, such that a 30-cycle PCR process also takes a substantial period of time to complete.
Various types of thermal cyclers based on the circulation of air have been proposed, such as described for example in U.S. Pat. Nos. 3,616,264, 4,420,679 and 5,455,175, but the previously known thermal cyclers of this type have not been found completely satisfactory for efficiently, rapidly and uniformly carrying out the PCR process, and/or do not allow standard sample holders of the rectangular matrix type, such as the microtiter plate, to be conveniently used.
An object of the present invention is to provide a novel method and apparatus for effecting rapid and uniform thermal cycling of samples, particularly a plurality of samples arranged in microtiter format. Another object of the invention is to provide a method and apparatus of the foregoing type particularly useful in the PCR process for DNA amplification and for cycle-sequencing.
According to one aspect of the present invention, there is provided a method of effecting rapid thermal cycling of samples, comprising: producing a high-velocity laminar air flow through a channel defining a closed loop flow path; energizing an electrical heater within the closed loop flow path to heat the air flowing therethrough to a desired temperature; introducing a sample holder containing at least one sample into a section of the closed loop flow path for exposing the sample holder to the high-velocity heated air flowing therethrough for rapidly heating the sample; and rapidly cooling the sample to a desired temperature by de-energizing the electrical heater, and opening an air outlet from the closed loop flow path, while continuing to produce the high-velocity air flow through the channel.
According to a further feature in the described preferred embodiment, the channel is of rectangular cross-section. This features aids in producing a uniform heating and/or cooling of the samples.
According to further preferred features, the channel defining the closed loop flow path is a closed loop channel having a selectively-openable inlet and a selectively-openable outlet.
Preferably, the air velocity is 10-30 m/sec at 25xc2x0 C. This has been found to produce laminar air flow with uniform heating/cooling of the samples in a microtiter-size sample holder.
According to further features in other described preferred embodiments, the section of the closed loop flowpath into which the sample holder is introduced is of decreasing cross-sectional area from the upstream side of the sample holder to the downstream side of the sample holder, to produce an increase in the velocity of the airflow at the downstream side as compared to that at the upstream side. Such an arrangement may be provided, for example, to compensate for the xe2x80x9cshading effectxe2x80x9d that may be produced with respect to the airflow from the upstream side to the downstream side of the sample holder where there is a relatively small spacing between the individual holders.
In one described embodiment, the decreasing cross-sectional area is effected by the provision of an inclined baffle in the section of the closed loop flow path into which the sample holder is introduced, the inclined baffle underlying the sample holder. In another described preferred embodiment, the decreasing cross-sectional area is effected by mounting the sample holder in an inclined position in the section of the closed loop flowpath.
According to a further feature in the preferred embodiment described below, the holder includes a cover containing another electrical heater which may be also energized during the thermal cycling process in order to prevent excess vaporization of the samples.
According to another aspect of the present invention, there is provided apparatus for effecting rapid thermal cycling of samples, comprising: a housing including a channel defining a closed loop flow path; an air impeller within the housing for producing a high-velocity laminar air flow through the closed loop flow path; an electrical heater within the housing for heating the air flowing through the closed loop flow path to a desired temperature; an access opening in a section of the channel for introducing a sample holder containing at least one sample into a sample compartment in the closed loop flow path for exposure to the high-velocity heated air flowing therethrough; and control means for selectively energizing the electrical heater to rapidly heat the sample by the heated air flowing through the closed loop flow path, and for selectively de-energizing the electrical heater and opening the closed loop flow path with respect to the atmosphere to rapidly cool the sample.
According to further features in the described preferred embodiment, the channel comprises a first section including first and second legs parallel to each other and joined by a first U-shaped juncture, and a second section including third and fourth legs parallel to each other and joined by a second U-shaped juncture; the first and second legs of the first section being perpendicularly joined to the third and fourth legs of the second section to define a closed loop flow path constituted of two U-shaped loops perpendicularly joined to each other. Such a folded construction provides a compact, space-saving arrangement for the closed loop flow path.
According to yet another aspect of the present invention, there is provided apparatus for effecting rapid thermal cycling of samples, comprising: a housing including a channel defining a closed loop flow path for air; an impeller within the housing for producing a high velocity air flow through the closed loop flow path; an electrical heater within the housing for heating the air flowing through the closed loop flow path to a desired temperature; an access opening in a section of the channel for introducing a sample holder containing at least one sample into a sample compartment in the closed loop flow path for exposure to the high-velocity heated air flowing therethrough; and a control means for selectively energizing the electrical heater to rapidly heat the sample by the heated air flowing through the closed loop flowpath, and for selectively de-energizing the electrical heater and opening the closed loop flow path with respect to the atmosphere to rapidly cool the sample; the section of the closed loop flow path into which the sample holder is introduced being of decreasing area from the upstream side of the sample holder to the downstream side of the sample holder, to produce an increase in the velocity of the airflow at the downstream side as compared to that at the upstream side.
Further features and advantages of the invention will be apparent from the description below.